A group of low-molecular-weight GTP-binding proteins (G-proteins) with molecular weights of 20,000-30,000 with no subunit structures is observed in organisms. To date, over fifty or more members have been found as the super family of the low-molecular-weight G-proteins in a variety of organisms, from yeast to mammals. The low-molecular-weight G-proteins are divided into four families of Ras, Rho, Rab and the others based on homologies of amino acid sequences. It has been revealed that the small G-proteins control a variety of cellular functions. For example, the Ras protein is considered to control cell proliferation and differentiation, and the Rho protein is considered to control cell morphological change, adhesion and motility.
The Rho protein, having GDP/GTP-binding activity and intrinsic GTPase activity, is believed to be involved in cytoskeletal responses to extracellular signals such as lysophosphatidic acid (LPA) and certain growth factors. When the inactive GDP-binding Rho is stimulated, it is transformed to the active GTP-binding Rho protein (hereinafter referred to as "the activated Rho protein") by GDP/GTP exchange proteins such as Smg GDS, Dbl or Ost. The activated Rho protein then acts on target proteins to form stress fibers and focal contacts, thus inducing the cell adhesion and motility (Experimental Medicine, Vol. 12, No. 8, 97-102 (1994); Takai, Y. et al., Trends Biochem. Sci., 20, 227-231 (1995)). On the other hand, the intrinsic GTPase activity of the Rho protein transforms the activated Rho protein to the GDP-binding Rho protein. This intrinsic GTPase activity is enhanced by what is called GTPase-activating proteins (GAP) (Lamarche, N. & Hall, A. et al., TIG, 10, 436-440 (1994)).
The Rho family proteins, including RhoA, RhoB, RhoC, Rac1, Rac2 and Cdc42, share more than 50% sequence identity with each other. The Rho family proteins are believed to be involved in inducing the formation of stress fibers and focal contacts in response to extracellular signals such as lysophosphatidic acid (LPA) and growth factors (A. J. Ridley & A. Hall, Cell, 70, 389-399 (1992); A. J. Ridley & A. Hall, EMBO J., 1353, 2600-2610 (1994)). The subfamily Rho is also considered to be implicated in physiological functions associated with cytoskeletal rearrangements, such as cell morphological change (H. F. Parterson et al., J. Cell Biol., 111, 1001-1007 (1990)), cell adhesion (Morii, N. et al., J. Biol. Chem., 267, 20921-20926 (1992); T. Tominaga et al., J. Cell Biol., 120, 1529-1537 (1993); Nusrat, A. et al., Proc. Natl. Acad. Sci. USA, 92, 10629-10633 (1995)*; Landanna, C. et al., Science, 271, 981-983 (1996)*, cell motility (K. Takaishi et al., Oncogene, 9, 273-279 (1994), and cytokinesis (K. Kishi et al., J. Cell Biol., 120, 1187-1195 (1993); I. Mabuchi et al., Zygote, 1, 325-331 (1993)). (An asterisk hereinafter indicates a publication issued after the first filed application which provides the right of the priority of the present application.) In addition, it has been suggested that the Rho is involved in the regulation of smooth muscle contraction (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); M. Noda et al., FEBS Lett., 367, 246-250 (1995); M. Gong et al., Proc. Natl. Acad. Sci. USA, 93, 1340-1345 (1996)*), and the expression of phosphatidylinositol 3-kinase (PI3 kinase) (J. Zhang et al., J. Biol. Chem., 268, 22251-22254 (1993)), phosphatidylinositol 4-phosphate 5-kinase (PI 4,5-kinase) (L. D. Chong et al., Cell, 79, 507-513 (1994)) and c-fos (C. S. Hill et al., Cell, 81, 1159-1170 (1995)).
Recently, it has also be found that Ras-dependent tumorigenesis is suppressed when the Rho protein of which the amino acid sequence has been partly substituted is introduced to cells, revealing that the Rho protein plays an important role in Ras-induced transformation, that is, tumorigenesis (G. C. Prendergast et al., Oncogene, 10, 2289-2296 (1995); Khosravi-Far, R. et al., Mol. Cell. Biol., 15, 6443-6453 (1995)*; R. Qiu et al., Proc. Natl. Acad. Sci. USA, 92, 11781-11785 (1995)*; Lebowitz, P. et al., Mol. Cell, Biol., 15, 6613-6622 (1995)*).
It has also been demonstrated that mutation of GDP/GTP-exchange proteins which act on the Rho protein results in cell transformation (Collard, J., Int. J. Oncol., 8, 131-138 (1996)*; Hart, M. et al., J. Biol. Chem., 269, 62-65 (1994); Horii, Y. et al., EMBO J., 13, 4776-4786 (1994)).
In addition, the Rho protein has been elucidated to be involved in cancer cell invasion, that is, metastasis (Yoshioka, K. et al., FEBS Lett., 372, 25-28 (1995)). The cancer cell invasion is closely dependent on changes in cancer cell activity to form cell adhesion. In this context, the Rho protein is also known to be involved in the formation of cell adhesion (see above Morii, N. et al. (1992); Tominaga, T. et al. (1993); Nusrat, A. et al. (1995); Landanna C. et al. (1996)*).
It has also been revealed that the Rho protein enhances not only cell proliferation, motility and aggregation, but also the contraction of smooth muscles. Recent studies have demonstrated that the Rho protein is involved in the contraction of smooth muscles (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); Noda, M. et al., FEBS Lett., 367, 246-250 (1995)). Therefore, it can reasonably be assumed that the activated Rho protein-binding proteins are also involved in the contraction of smooth muscles.
These findings indicate that the Rho protein controls a variety of signal transduction pathways for cell morphological change, adhesion, motility, cytokinesis, tumorigenesis, metastasis, vascular smooth muscle contraction, etc. The Rho protein acts on a number of target molecules to control signal transduction pathways.
It is only recently (after the first filed application which provides the right of the priority of the present application) that a several proteins have been reported as candidates of Rho-targets in mammals: protein kinase N (PKN) (Watanabe, G. et al., Science, 271, 645-648 (1996)*; Amano, M. et al., Science, 271, 648-650 (1996)*), rhophilin (Watanabe, G. et al., Science, 271, 645-648 (1996)*, citron (Madaule, P. et al., FEBS Lett., 377, 243-248 (1995)*), ROK.alpha. (Leung, T. et al., J. Biol. Chem., 270, 29051-29054 (1995)*), Rho-binding kinase (Matsui, T. et al., EMBO J., 15, 2208-2216 (1996)*) and rhotekin (Reid, T. et al., J. Biol. Chem., 271, 13556-13560 (1996)*). All these proteins bind to GTP-binding RhoA protein, except that citron binds also to GTP-binding Racl.
Among these proteins, PKN has an enzymatic region which closely resembles the protein kinase region of protein kinase C and exhibits serine/threonine kinase activity (Mukai, H. & Ono, Y., Biochem. Biopys. Res. Commun., 199, 897-904 (1994); Mukai, H. et al., Biochem. Biopys. Res. Commun., 204, 348-356 (1994)). On the other hand, ROK.alpha. and Rho-binding kinase (Matsui, T. et al. (1996)*, ibid.) also have amino acid sequences resembling a serine/threonine kinase region (Leung, T. et al. (1995)*, ibid.).
In addition to those reported in mammals, protein kinase C1 (PKC1) in yeast (Saccharomyces cerevisiae) has recently been identified as a target protein of the Rho1 protein, corresponding to RhoA in mammals (Nonaka, H. et al., EMBO J., 14, 5931-5938 (1995)*). Only recently, 1,3-.beta.-glucan synthesizing enzyme has been identified as a target protein of the Rholp protein in yeast (Saccharomyces cerevisiae) (Drgonova, J. et al., Science, 272, 277-279 (1996)*; Qadota, H. et al., Science, 272, 279-281 (1996)*).
However, mechanisms of intercellular signal transduction involving the activated Rho protein, particularly those of tumorigenesis and smooth muscle contraction, are still unknown.